Abstract
Fluorescent in situ hybridization (FISH) provides sensitive detection and visualization of RNA transcripts in tissues and cells with high resolution. We present here a multiplex RNA FISH method using enhanced tyramide signal amplification (TSA) for colocalization analysis of three different transcripts in intact zebrafish brains. To achieve enhancement of fluorescent signals, essential steps of the FISH procedure are optimized including embryo permeability, hybridization efficacy, and fluorogenic TSA-reaction conditions. Critical to this protocol, the enzymatic peroxidase (PO) reactivity is significantly improved by the application of viscosity-increasing polymers, PO accelerators, and highly effective bench-made tyramide substrates. These advancements lead to an optimized TSA–FISH protocol with dramatically increased signal intensity and signal-to-background ratio allowing for visualization of three mRNA transcript patterns simultaneously. The TSA–FISH procedure can be combined with immunofluorescence (IF) to compare mRNA transcript and protein expression patterns.
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References
Femino A, Fay FS, Fogarty K, Singer RH (1998) Visualization of single RNA transcripts in situ. Science 280(5363):585–590
Raj A, van den Bogaard P, Rifkin SA, van Oudenaarden A, Tyagi S (2008) Imaging individual mRNA molecules using multiple singly labeled probes. Nat Methods 5(10):877–879
Hauptmann G, Lauter G, Söll I (2016) Detection and signal amplification in zebrafish RNA FISH. Methods 98:50–59. https://doi.org/10.1016/j.ymeth.2016.01.012
Player AN, Shen LP, Kenny D, Antao VP, Kolberg JA (2001) Single-copy gene detection using branched DNA (bDNA) in situ hybridization. J Histochem Cytochem 49(5):603–611
Choi HM, Chang JY, Trinh le A, Padilla JE, Fraser SE, Pierce NA (2010) Programmable in situ amplification for multiplexed imaging of mRNA expression. Nat Biotechnol 28(11):1208–1212. https://doi.org/10.1038/nbt.1692. [pii] nbt.1692
Choi HMT, Beck VA, Pierce NA (2014) Next-generation in situ hybridization chain reaction: higher gain, lower cost, greater durability. ACS Nano 8(5):4284–4294
Speel EJ (1999) Robert Feulgen Prize Lecture 1999. Detection and amplification systems for sensitive, multiple-target DNA and RNA in situ hybridization: looking inside cells with a spectrum of colors. Histochem Cell Biol 112(2):89–113
Speel EJ, Ramaekers FC, Hopman AH (1995) Cytochemical detection systems for in situ hybridization, and the combination with immunocytochemistry, ‘who is still afraid of red, green and blue?’. Histochem J 27(11):833–858
Bobrow MN, Harris TD, Shaughnessy KJ, Litt GJ (1989) Catalyzed reporter deposition, a novel method of signal amplification. Application to immunoassays. J Immunol Methods 125(1–2):279–285. [pii] 0022-1759(89)90104-X
Speel EJM, Hopman AHN, Komminoth P (1999) Amplification methods to increase the sensitivity of in situ hybridization: play CARD(S). J Histochem Cytochem 47(3):281–288
Kosman D, Mizutani CM, Lemons D, Cox WG, McGinnis W, Bier E (2004) Multiplex detection of RNA expression in Drosophila embryos. Science 305(5685):846. https://doi.org/10.1126/science.1099247. [pii] 305/5685/846
Tessmar-Raible K, Steinmetz PR, Snyman H, Hassel M, Arendt D (2005) Fluorescent two-color whole mount in situ hybridization in Platynereis dumerilii (Polychaeta, Annelida), an emerging marine molecular model for evolution and development. BioTechniques 39(4):460, 462, 464. doi:000112023 [pii]
Clay H, Ramakrishnan L (2005) Multiplex fluorescent in situ hybridization in zebrafish embryos using tyramide signal amplification. Zebrafish 2(2):105–111. https://doi.org/10.1089/zeb.2005.2.105
Lauter G, Söll I, Hauptmann G (2011) Multicolor fluorescent in situ hybridization to define abutting and overlapping gene expression in the embryonic zebrafish brain. Neural Dev 6(1):10. https://doi.org/10.1186/1749-8104-6-10. [pii] 1749-8104-6-10
Vize PD, McCoy KE, Zhou X (2009) Multichannel wholemount fluorescent and fluorescent/chromogenic in situ hybridization in Xenopus embryos. Nat Protoc 4(6):975–983. https://doi.org/10.1038/nprot.2009.69. [pii] nprot.2009.69
Denkers N, Garcia-Villalba P, Rodesch CK, Nielson KR, Mauch TJ (2004) FISHing for chick genes: triple-label whole-mount fluorescence in situ hybridization detects simultaneous and overlapping gene expression in avian embryos. Dev Dyn 229(3):651–657. https://doi.org/10.1002/dvdy.20005
Lecuyer E, Yoshida H, Parthasarathy N, Alm C, Babak T, Cerovina T, Hughes TR, Tomancak P, Krause HM (2007) Global analysis of mRNA localization reveals a prominent role in organizing cellular architecture and function. Cell 131(1):174–187. https://doi.org/10.1016/j.cell.2007.08.003. [pii] S0092-8674(07)01022-7
Lauter G, Söll I, Hauptmann G (2013) Molecular characterization of prosomeric and intraprosomeric subdivisions of the embryonic zebrafish diencephalon. J Comp Neurol 521(5):1093–1118. https://doi.org/10.1002/cne.23221
Herget U, Ryu S (2015) Coexpression analysis of nine neuropeptides in the neurosecretory preoptic area of larval zebrafish. Front Neuroanat 9:2. https://doi.org/10.3389/fnana.2015.00002
Hauptmann G, Gerster T (2000) Regulatory gene expression patterns reveal transverse and longitudinal subdivisions of the embryonic zebrafish forebrain. Mech Dev 91(1–2):105–118. [pii] S0925-4773(99)00277-4
Hauptmann G, Söll I, Gerster T (2002) The early embryonic zebrafish forebrain is subdivided into molecularly distinct transverse and longitudinal domains. Brain Res Bull 57(3–4):371–375. S0361923001006918 [pii]
Affaticati P, Yamamoto K, Rizzi B, Bureau C, Peyrieras N, Pasqualini C, Demarque M, Vernier P (2015) Identification of the optic recess region as a morphogenetic entity in the zebrafish forebrain. Sci Rep 5:8738. https://doi.org/10.1038/srep08738
Tautz D, Pfeifle C (1989) A non-radioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals translational control of the segmentation gene hunchback. Chromosoma 98(2):81–85
Hauptmann G (1999) Two-color detection of mRNA transcript localizations in fish and fly embryos using alkaline phosphatase and beta-galactosidase conjugated antibodies. Dev Genes Evol 209(5):317–321
Hauptmann G (2001) One-, two-, and three-color whole-mount in situ hybridization to Drosophila embryos. Methods 23(4):359–372. https://doi.org/10.1006/meth.2000.1148. [pii] S1046-2023(00)91148-4
Hauptmann G, Gerster T (1994) Two-color whole-mount in situ hybridization to vertebrate and Drosophila embryos. Trends Genet 10(8):266
Hauptmann G, Gerster T (1996) Multicolour whole-mount in situ hybridization to Drosophila embryos. Dev Genes Evol 206(4):292–295. https://doi.org/10.1007/s004270050055
Jowett T, Lettice L (1994) Whole-mount in situ hybridizations on zebrafish embryos using a mixture of digoxigenin- and fluorescein-labelled probes. Trends Genet 10(3):73–74
O’Neill JW, Bier E (1994) Double-label in situ hybridization using biotin and digoxigenin-tagged RNA probes. BioTechniques 17(5):870, 874-875
Hauptmann G, Söll I, Krautz R, Theopold U (2016) Multi-target chromogenic whole-mount in situ hybridization for comparing gene expression domains in drosophila embryos. J Vis Exp (107). https://doi.org/10.3791/53830
Söll I, Hauptmann G (2015) Multicolored visualization of transcript distributions in Drosophila embryos. NeuroMethods 99:45–59. https://doi.org/10.1007/978-1-4939-2303-8_3
Lauter G, Söll I, Hauptmann G (2011) Two-color fluorescent in situ hybridization in the embryonic zebrafish brain using differential detection systems. BMC Dev Biol 11:43. https://doi.org/10.1186/1471-213X-11-43
Lauter G, Söll I, Hauptmann G (2014) Sensitive whole-mount fluorescent in situ hybridization in zebrafish using enhanced tyramide signal amplification. Methods Mol Biol 1082:175–185. https://doi.org/10.1007/978-1-62703-655-9_12
Hauptmann G, Lauter G, Söll I (2015) Application of alkaline phosphatase-mediated azo dye staining for dual fluorescent in situ hybridization in zebrafish. NeuroMethods 99:393–404. https://doi.org/10.1007/978-1-4939-2303-8_20
Hauptmann G, Gerster T (2000) Multicolor whole-mount in situ hybridization. Methods Mol Biol 137:139–148. https://doi.org/10.1385/1-59259-066-7:139. [pii] 1-59259-066-7-139
Fischer AH, Jacobson KA, Rose J, Zeller R (2008) Media for mounting fixed cells on microscope slides. CSH Protoc 2008:pdb ip52
Liu G, Amin S, Okuhama NN, Liao G, Mingle LA (2006) A quantitative evaluation of peroxidase inhibitors for tyramide signal amplification mediated cytochemistry and histochemistry. Histochem Cell Biol 126(2):283–291. https://doi.org/10.1007/s00418-006-0161-x
Acknowledgments
Imaging was in part performed at the Live Cell Imaging unit, Department of Biosciences and Nutrition, Karolinska Institutet, Huddinge, Sweden, supported by grants from the Knut and Alice Wallenberg Foundation, the Swedish Research Council, and the Centre for Biosciences.
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Lauter, G., Söll, I., Hauptmann, G. (2020). Sensitive Multiplexed Fluorescent In Situ Hybridization Using Enhanced Tyramide Signal Amplification and Its Combination with Immunofluorescent Protein Visualization in Zebrafish. In: Sprecher, S. (eds) Brain Development. Methods in Molecular Biology, vol 2047. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9732-9_22
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DOI: https://doi.org/10.1007/978-1-4939-9732-9_22
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